Bacterial strain, processed plant extracts, compositions containing same, processes for their preparation and their therapeutic and industrial applications

ABSTRACT

The present invention discloses: (i) a non-pathogenic probiotic microorganism and its probiotic/therapeutic uses; (ii) a formulation comprising an. aqueous solution of a volatile fraction (VF) prepared from the extract of at least one plant derived material and its therapeutic uses; (iii) a process of manufacturing the formulation from the plant derived material; (iv) a probiotic composition comprising the non-pathogenic probiotic microorganism of the invention and/or other probiotic microorganism(s) and the formulation of the invention, and its probiotic/therapeutic uses; (v) a composition for industrial applications comprising the formulation of the invention and microorganism(s) of industrial applicability; and (vi) industrial processes and apparatuses in which the latter composition is used.

This is a Divisional Application of U.S. application Ser. No. 09/725,846filed Nov. 30, 2000, which is pending, which is a Continuation-in-Partof PCT application Ser. No. IL00/00318, filed Jun. 1, 2000.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to (i) a non-pathogenic probioticmicroorganism and its probiotic/therapeutic uses; (ii) a formulationcomprising an aqueous solution of a volatile fraction (VF) prepared fromthe extract of at least one plant derived material and its therapeuticuses; (iii) a process of manufacturing the formulation from the plantderived material; (iv) a probiotic composition comprising thenon-pathogenic probiotic microorganism of the invention and/or otherprobiotic microorganism(s) and the formulation of the invention, and itsprobiotic/therapeutic uses; (v) a composition for industrialapplications comprising the formulation of the invention andmicroorganism(s) of industrial applicability; and (vi) industrialprocesses and apparatuses in which the latter composition is used.

Under normal conditions, the gastrointestinal (GI) tract microfloracontributes significantly to the health and well being of an individual.It is well known that the microflora is a complex and diverse populationof microorganisms, which may have both beneficial and harmful effects onthe individual. Under normal gestation conditions, the fetus in utero issterile, but on passage through the vagina during birth it acquiresmicroorganisms resulting in the formation of a gut microflora. The finalindigenous gut microflora which stabilizes in the gut is a very complexcollection of over one thousand different types of microorganisms,consisting of about 400 different types of bacteria [Fuller R. J.Applied Bacteriology 66:365-378 (1989)]. The composition of the gutmicroflora is determined by both host and microbial factors, andalthough there are a lot of bacteria which can survive and grow in theGI tract, there are many which cannot. In addition, the survivingorganisms have to avoid the effect of peristalsis which normally flushesout the bacteria with the food. This may be achieved by the bacteriaimmobilizing themselves by attachment to the gut wall, and/or bymultiplying at a rate that exceeds the rate of removal by peristalsis.

In general, the presence of gut microflora is symbiotic as themicroflora not only assists in disintegrating some undigestable foods,it also protects the individual from infections caused by pathogens.This latter phenomenon has been described under such names as ‘bacterialantagonism’, ‘bacterial interference’, ‘barrier effect’, ‘colonizationresistance’, ‘competitive exclusion’ and many others.

The gut protective microflora is very stable. However, it is lesseffective in the young, elderly and the compromised patient. Further, itcan be influenced by certain dietary and environmental factors, thethree most important being excessive hygiene, antibiotic therapy andstress.

Under conditions where the balance of the gut microflora is adverselyaffected, probiotics become of potential value in restoring the gutmicroflora and enabling the individual host to return to normal.

Probiotics are a class of microorganisms defined as live microbialorganisms that beneficially affect the animal and human hosts. Thebeneficial effects include improvement of the microbial balance of theintestinal microflora or improving the properties of the indigenousmicroflora. The beneficial effects of probiotics may be mediated by adirect antagonistic effect against specific groups of organisms,resulting in a decrease in numbers, by an effect on their metabolism orby stimulation of immunity. Probiotics may suppress viable counts of anundesired organism by producing antibacterial compounds, by competingfor nutrients or for adhesion sites. Further, they may alter microbialmetabolism by increasing or decreasing enzyme activity or they maystimulate the immune system by increasing antibody levels or increasingmacrophage activity.

WO95/16461 describes a probiotic composition of anaerobic bacteriaeffective in controlling or inhibiting Salmonella colonization indomesticated animals. The probiotic composition includes populations orcultures of 29 substantially biologically pure bacteria, inter alia, E.coli. However, the suppression of the pathogen by the probioticcomposition described in this PCT publication requires the combinedaction of a large number of bacterial strains.

WO97/35596 describes the administration of a freshly prepared probioticmixture obtained by mixing a powder containing Lactobacillus reuteri,Lactobacillus acidophilus and Bifidobacterium infantis with a liquid.The mixture is described to be effective in preventing infectiousdiarrhea or diarrhea caused by antibiotic therapy in humans. Thefreeze-dried live bacteria are, however, in anabiotic state. The need towet the microorganism before administration, in order to reinstate itsvitality, is a disadvantage, since normally many bacteria do not survivethe re-hydration. Moreover, the surviving organisms are not immediatelymetabolically active, and cannot survive the extreme, acidic conditionsof the stomach. Furthermore, when administered to a recipient withdiarrhea, the rate of their removal from the gut may exceed the rate ofreinstation of viability, resulting in minimal or no beneficiary effect.

Preservation of viability and conservation of the activity of probioticorganisms by their formulation is the issue of numerous publications.WO98/26787 describes the enhancement of a resident population of lacticacid-producing microorganisms, preferably lactobaccillii, in the GItract of an animal by providing the same with β-glucan, optionally incombination with prebiotic and/or probiotic microorganisms.

WO97/34591 also describes the-enhancement of resident population ofmicroorganisms, or the suppression of the undesired resident populationat a selected site of the GI tract of an individual, by providing theindividual with a selected modified or unmodified starch or mixturesthereof, which act as carrier for one or more probiotic microorganismsand as a growth or maintenance medium for the microorganisms. Theprobiotic elements are bound to the carrier in a manner so as to protectthe microorganisms during passage to the large bowel or other regions ofthe GI tract.

Microorganisms are used in a variety of industrial processes. Theseprocesses can be divided into continuous processes and batch processes.In continuous processes, such as a continuous aerobic or anaerobicfermentation line, used for the production of a variety of fermentationproducts, including, for example, ethanol and methanol, a fermentableraw material is continuously fed into the line and the fermentiveproduct is continuously collected from the line, in a controlledfashion, such that the microorganism population in the line is sustainedthroughout the process. In batch processes, such as, but not limited to,batch aerobic or anaerobic fermentation, biodegradation of oil in, forexample, oil spills, and the like, a fermentable raw material is mixedwith a population of microorganism(s) so as to instate fermentation.Both in continuous fermentation and in batch fermentation processes, itis desired, in some cases, to start or reinstate the process with alarge and viable population of the relevant microorganism(s). Forexample, when biodegrading oil, it is advantageous to start the processwith an as large as possible population of the microorganismsPseudomonas spp. or Alcaligenes spp., for example, so as to minimize thetime for complete biodegradation of the oil. When using filters enrichedwith microorganism(s) for biodegradation of organic fumes or volatiles,the result is a continuous process in which polluting compounds aredegraded into harmless compounds. However, the microorganisms present inthe filter depend for their survival on continuous supply of organicfumes or volatiles. Nevertheless, in some instances the supply oforganic fumes or volatiles is discontinued and, as a result, themicroorganism(s) are lost. Under such circumstances, rein station of aneffective viable amount of microorganism(s) in the filter is required ina short period of time, so as permit immediate restoration of afunctional filter.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a formulation in which microorganisms, includingprobiotic microorganisms and microorganisms useful in a variety ofindustrial applications, could be maintained viable and in ametabolically active form for long periods of time.

While reducing the present invention to practice, it has beenunexpectedly found that a single species of a non-pathogenic probioticmicroorganism derived from E. coli is, alone, capable of restoringnormal GI flora of man and of a variety of mammals and avians. It hasalso been surprisingly found that this microorganism, as well as othermicroorganisms, could be preserved for long periods of time, in a viableand metabolically active form, in a formulation comprising watersolution of volatile fraction(s) of various plant extracts. A probioticcomposition comprising the probiotic organism suspended in theformulation was found to be effective in the treatment and prevention ofvarious gastrointestinal disorders. It has further been unexpectedlyfound that the formulation per se is effective as a body weight gainenhancer and as an immuno-stimulator in mammals and avians.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anEscherichia coli strain BU-230-98 ATCC Deposit No. 20226 (DSM 12799).

According to another aspect of the present invention there is provided aprobiotic composition comprising viable Escherichia coli strainBU-230-98 ATCC Deposit No. 20226 (DSM 12799) and a formulation formaintaining viability of the Escherichia coli strain. The probioticcomposition preferably further comprising at least one flavoring agent.Preferably, the formulation includes at least one volatile fraction (VF)of a plant extract prepared by steam distillation of a plant (e.g.,beet, dill, parsley or grapefruit) extract under reduced pressure and ata bath temperature preferably not exceeding 38° C.

The probiotic composition of the present invention may be identified forpreventing or treating gastro-enteric infections or disorders,maintaining or reinstating normal gastro-intestinal microflora,preventing or treating diarrhea, preventing or treating gastro-entericinfection caused by an enteric pathogen, such as a Gram negativebacterium or Gram positive bacterium, preventing or treatinggastro-enteric Salmonella infection, preventing or treating infectiousdiarrhea, caused by, for example C. difficile, Salmonella, particularlyS. Shigella, Campylobacter, E. coli, Proteus, Pseudomonas orClostridium, chronic diarrhea or diarrhea resulting from antibiotictherapy, radiotherapy or chemotherapy, and/or for normalizing thephysiological activity of the gastrointestinal tract.

According to yet another aspect of the present invention there isprovided a formulation comprising at least one volatile fraction (VF) ofa plant extract, the volatile fraction is prepared by steam distillationof the plant extract under reduced pressure and at a bath temperaturenot exceeding 38° C. The plant extract is preferably obtainable from aplant organ selected from the group consisting of leaves, stems, rootsand fruit. The plant can be a vegetable, such as soy bean, alfalfa,garlic, beet and cabbage, or a herb, such as parsley, mint and dill. Theformulation may further include a beehive product, such as, but notlimited to, propolis. The formulation can serve as a veterinary feed orfood additive for enhancing animal weight gain in a recipient. It canalso serve to preserve viable bacteria as is further described herein.

Hence, according to an additional aspect of the present invention thereis provided a process of preparing a volatile fraction of a plant,comprising the steps of (a) grinding a plant derived material to give aplant biomass; (b) mixing the plant biomass with water and stirring atambient temperature; (c) steam distilling the mixture obtained in step(b) under reduced pressure and at a bath temperature not exceeding 38°C.; and (d) collecting volatile fraction obtained from the steamdistillation. Preferably, the reduced pressure is of 5-10 mbar. Thisformulation can be used as a food additive, a feed additive and hasunexpected therapeutic uses, as well as advantageous bacteriapreservation properties. A therapeutic formulation containing thevolatile fraction of a plant is also disclosed and may be prepared bymixing at least one volatile fraction (VF) of a plant extract withphysiologically or veterinary acceptable additives, carriers ordiluents. Preferably, the volatile fraction is prepared by steamdistillation of the plant extract under reduced pressure and at a bathtemperature not exceeding 38° C.

Thus, according to still another aspect of the present invention thereis provided a probiotic composition comprising (a) an effective amountof at least one viable probiotic microorganism having a beneficialbiological or therapeutic activity in the gastrointestinal tract; and(b) at least one volatile fraction (VF) of a plant extract preferablyprepared as described herein. The probiotic microorganism can be E.coli, preferably, strain BU-230-98, ATCC Deposit No. 202226 and it canbe identified for treatment or prevention of any of the abovedisorders/pathologies/diseases/syndromes and in addition or as analternative in can be identified for treatment or prevention ofdyspeptic symptoms, for enhancing the immune response in a patientsuffering from an immune disorder, resulting from, for example,immune-response suppression therapy.

According to still another aspect of the present invention there isprovided a composition comprising (a) at least one microorganism, suchas, Escherichia spp., Alcaligenes spp., Arthrobacter spp.,Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonasspp. and Pseudomonas spp.; and (b) at least one volatile fraction (VF)of a plant extract, the at least one volatile fraction is selected so asto sustain viability of the at least one microorganism for at least 2months, preferably at least 3-6 months, more preferably 6-12 months ormore at room temperature.

According to another aspect of the present invention there is provided aprocess of growing Escherichia coli strain BU-230-98 ATCC Deposit No.20226 (DSM 12799), the process comprising the step of seeding a startercloture of Escherichia coli strain BU-230-98 ATCC Deposit No. 20226 (DSM12799) into a growth media and growing the Escherichia coil strain up tooptical density of 15-30 at 650 nm.

According to yet another aspect of the present invention there isprovided a process of manufacturing a probiotic composition, the processcomprising the step of suspending viable Escherichia coli strainBU-230-98 ATCC Deposit No. 20226 (DSM 12799) in a formulation formaintaining viability of the Escherichia coli strain. Preferably, theformulation includes at least one volatile fraction (VF) of a plantextract, prepared, preferably by steam distillation of the plant extractunder reduced pressure and at a bath temperature not exceeding 38° C.,obtained from, for example, a plant organ selected from the groupconsisting of leaves, stems, roots and fruit, either a vegetable and aherb, such as soy bean, parsley, mint, dill, alfalfa, garlic, beet orcabbage.

According to still another aspect of the present invention there isprovided a process of preparing a probiotic composition, the processcomprising the step of suspending an effective amount of at least oneviable probiotic microorganism having a beneficial biological ortherapeutic activity in the gastrointestinal tract in an formulationcontaining at least one volatile fraction (VF) of a plant extract.

According to an additional aspect of the present invention there isprovided a process of preparing a composition including viablemicroorganisms, the process comprising the step of suspending at leastone microorganism in a formulation containing at least one volatilefraction (VF) of a plant extract, the at least one volatile fraction isselected so as to sustain viability of the at least one microorganismfor at least 2 months at room temperature. The microorganism can be, forexample, Escherichia spp., Alcaligenes spp., Arthrobacter spp.,Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonasspp. or Pseudomonas spp.

According to yet an additional aspect of the present invention there isprovided a dispenser of microorganisms comprising a reservoir and adispensing mechanism being connected thereto, the reservoir housing atleast one microorganism in a formulation containing at least onevolatile fraction (VF) of a plant extract, the at least one volatilefraction is selected so as to sustain viability of the at least onemicroorganism for at least 2 months at room temperature.

According to still an additional aspect of the present invention thereis provided a method of biocontrol of an organism, the method comprisingthe step of spreading in an endemic area at least one viablemicroorganism capable of biocontrolling the organism, the at least oneviable microorganism being in a formulation containing at least onevolatile fraction (VF) of a plant extract, the at least one volatilefraction is selected so as to sustain viability of the at least onemicroorganism for at least 2 months at room temperature.

According to a further aspect of the present invention there is provideda method of eliminating an oil pollution, the method comprising the stepof spreading in a polluted area at least one viable microorganismcapable of degrading oil, the at least one viable microorganism being ina formulation containing at least one volatile fraction (VF) of a plantextract, the at least one volatile fraction is selected so as to sustainviability of the at least one microorganism for at least 2 months atroom temperature.

According to yet a further aspect of the present invention there isprovided a method of restoring organic fumes degrading microorganismpopulation in a biofilter, the method comprising the step of dispensingonto the filter at least one viable microorganism capable of degradingorganic fumes, the at least one viable microorganism being in aformulation containing at least one volatile fraction (VF) of a plantextract, the at least one volatile fraction is selected so as to sustainviability of the at least one microorganism for at least 2 months atroom temperature.

According to still a further aspect of the present invention there isprovided a method of preparing a starter for a fermentation process, themethod comprising the step of growing a sufficient amount of a startermicroorganism and suspending the starter microorganism in a formulationcontaining at least one volatile fraction (VF) of a plant extract, theat least one volatile fraction is selected so as to sustain viability ofthe starter microorganism for at least 2 months at room temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic depiction of a dispenser according to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is of (i) a non-pathogenic probiotic microorganismand its probiotic/therapeutic uses; (ii) a formulation comprising anaqueous solution of a volatile fraction (VF) prepared from the extractof at least one plant derived material and its therapeutic uses; (iii) aprocess of manufacturing the formulation from the plant derivedmaterial; (iv) a probiotic composition comprising the non-pathogenicprobiotic microorganism of the invention and/or other probioticmicroorganism(s) and the formulation of the invention, and itsprobiotic/therapeutic uses; (v) a composition for industrialapplications comprising the formulation of the invention andmicroorganism(s) of industrial applicability; and (vi) industrialprocesses and apparatuses in which the latter composition is used.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

In one aspect, the present invention relates to a probioticmicroorganism being a non-pathogenic bacterium derived from E. coli,having a beneficial physiological and/or therapeutic activity in, forexample, the GI tract and deposited at the ATCC under deposit No. 202226and at DSMZ under deposit No. 12799.

“Probiotic” is used herein as an adjective to describe an isolatedbacteria having the property of inhibiting the growth of at least onepathogen. The test of an inhibition used herein was an in vitro test onsolid medium in which culture supernatants of candidate isolatedbacteria were observed for their property of inhibiting the growth of apathogen when applied to the surface of the solid medium. Typically, apaper disc impregnated with the culture supernatant of a candidateprobiotic strain was placed on the surface of an agar plate seeded withthe pathogen. Probiotic bacterial supernatants caused a ring of clearagar or of reduced growth density indicating inhibition of the pathogenin the vicinity of the disc. There are other tests for inhibition whichare available or could be devised, including direct growth competitiontests, in vitro or in vivo which can generate a panel of probioticbacteria similar to that described herein. The bacterial strainsidentified by any such test are within the category of probioticbacteria, as the term is used herein.

The probiotic bacteria of the present invention may serve as a food orfeed additive, so as to provide food or feed product supplemented withviable Escherichia coli strain BU-230-98 ATCC Deposit No. 20226 (DSM12799). A food product of the present invention may further include atleast one milk derived substance and may be selected from the groupconsisting of a cheese and a yogurt.

The present invention further relates to a formulation comprising atleast one volatile fraction (VF) of a plant extract, the volatilefraction being prepared by distillation of the plant extract underreduced pressure and at a bath temperature typically not exceeding 38°C.

The plant matter from which the volatile fraction may be obtained may beany suitable plant part such as fruit, leaf, stem or root. Many plantsare suitable as a source for the volatile fractions, for example apple,citrus, soy bean, beet, cabbage, garlic and. alfalfa, as well as herbssuch as parsley, mint and dill. The formulations of the invention mayoptionally further comprise a suitable amount of a volatile fraction ofan apicultural product such as honey, propolis or other beehive product,which may be prepared in the same manner as described herein forobtaining volatile fractions from plant extracts.

As will be described in more detail in the following Examples, theformulation itself may be used as a food/feed additive. It has beenfound by the inventors to have a weight gain enhancing activity and itconstitutes an aspect of the invention. According to a particularembodiment of this aspect of the invention, the animal weight gainenhancing formulation or feed additive comprises distilled water andvolatile fractions of alfalfa, soy beans, beet and dill, preferably at avolume ratio of the volatile fractions of 2:8:1:4. Alternatively, aformulation or feed additive of the present invention comprisesdistilled water and volatile fractions of carrot, beet, dill andgrapefruit (1 part each) per 100 parts of water. Still alternatively, aformulation or food additive of the present invention comprisesdistilled water and volatile fractions of beet, dill and parsley (1 parteach) per 100 parts of water.

In a further aspect, the invention relates to a composition comprisingthe above formulation of the invention and at least one viablemicroorganism, either a probiotic organism having a therapeutic orbeneficial biological activity in, for example, the GI tract of humanand/or animal, to thereby provide a probiotic composition, or anorganism having industrial applications, to thereby provide acomposition of industrial applicability.

A particular advantage of the probiotic and industrial compositions ofthe invention stems from the fact that they are liquid preparations.Being under biologically active conditions, the formulation serves alsoas a supportive medium for living bacteria, as opposed to lyophilizedformulations where the bacteria are in an anabiotic condition. As aresult, the probiotic composition of the invention, for example, isactive immediately following oral administration, beginning with theupper portion of the GI tract, where primary effects of the majority ofintestinal pathogens take place, causing development of adversegastro-enteric syndromes. The probiotic compositions of the inventionmay also be used as body weight-increasing preparations or food/feedadditives. Similarly, the industrial composition of the invention can beused to efficiently reinstate a population of microorganisms ofindustrial applicability.

Thus, it is noted that, for probiotic applications, probiotic bacteriaother than such belonging to the strain BU-230-98 (ATCC Deposit No.202226, also deposited at the DSM under No. 12799), may be used with theplant material derived formulation of the invention. Such probioticbacteria have a very broad spectrum of antagonistic activity. They alsobelong to the same phylogenetic group of the majority of intestinalpathogens and share the same systems of survival. Therefore, thesuppression and exclusion of intestinal pathogens may include manydifferent. mechanisms, for example, secretion of antagonistic material,competition for utilization of nutrients and competition for adhesionreceptors. Thus, any non-pathogenic bacteria which comply with thesecriteria, may be used in the probiotic compositions of the invention.

According to a further aspect of the invention, the probioticcompositions of the invention may be used for preventing or treatinggastro-enteric infections. Term ‘gastro-enteric infection ’ is to betaken to mean any infection caused an enteric pathogen, including, interalia, Gram negative and Gram positive bacteria. By improving the generalbalance and health of the GI tract, the formulations and probioticcompositions of the invention may be instrumental in prophylaxis of alsoGI infections caused by yeast, viruses and protozoa.

The term ‘therapeutically effective amount’ or ‘effective amount’ forpurposes herein is the amount determined by such considerations as areknown in the art. The amount must be sufficient to enable the efficientrestoration of the GI microflora thus leading to the normalization ofthe function of the GI tract.

In a particular embodiment, the probiotic composition for preventing thedevelopment of gastro-enteric infections comprises the formulation ofthe invention made from distilled water and volatile fractions ofalfalfa, soy beans, beet, dill and mint as defined herein, at a volumeratio of, e.g., 5:1:5:15: 1, and a therapeutically effective amount of aprobiotic bacteria such as E. coli ATCC Deposit No. 202226 (identicalwith DSM 12799) or any other probiotic bacteria.

A specific example for an gastro-enteric infection is that caused bySalmonella and the invention is of particular advantage in preventing ortreating gastro-enteric infections caused thereby.

Further, the food additive or formulation of the invention, alone or incombination with an effective amount of a probiotic microorganism, suchas the E. coli strain ATCC Deposit No. 202226 (identical with DSM12799), may be used for treating or preventing infectious diarrhea,chronic diarrhea or diarrhea caused by antibiotic or chemotherapy.

According to a further specific embodiment of the invention, such aprobiotic composition for treating diarrhea may comprise distilled waterand volatile fractions of alfalfa, soy beans, beet, dill and mint at avolume ratio of, e.g., 5:1:5:15:1, and a therapeutically effectiveamount of the ATCC No. 202226 (DSMZ 12799).

The infectious diarrhea may be caused by numerous factors, for example,by a microorganism selected from C. difficile, Salmonella, particularlyS. Shigella, Campylobacter, E. coli, Proteus, Pseudomonas, Clostridium,enteric Staphylococcus. These are but few of many infecting agents.

Yet further, the probiotic compositions of the invention may be used foreffectively restoring the GI microflora in a subject in need of suchtreatment which leads to the normalization of the function of the GItract. Such compositions may include, for example, distilled water andvolatile fractions of alfalfa, soy bean, beet, dill, mint, parsley andcabbage, preferably at a ratio of volatile fractions of, e.g.,1:5:5:2:2:1, and a therapeutically effective amount of the probioticbacteria ATCC Deposit No. 202226 (DSM 12799).

Other purposes for which the probiotic compositions of the invention,comprising at least one probiotic bacteria having a therapeutic effectin, for example, the GI tract, include alleviation of lactoseintolerance in subjects suffering from lactose intolerance, treatment ofenterocolitis, treatment of constipation, for reduction of cholesterollevels in the blood, for treatment of dyspeptic symptoms, and/or forstimulation of the immune system in subjects suffering from an immunesystem disorder, which may be an immune disorder caused byimmuno-suppressive therapy.

In a different aspect, the invention relates to a process for preparinga volatile fraction of a plant extract, which process comprises thesteps of: (a) grinding plant derived matter to obtain a plant biomass;(b) mixing the plant biomass obtained in step (a) with water at a weightproportion of preferably 3 parts water to 1 part of the plant biomassand stirring the same for at least 2 hours at ambient temperature; (c)distilling the mixture obtained in step (b) under reduced pressure andat a bath temperature preferably not exceeding 38° C.; and (d)collecting the volatile fraction obtained from the steam distillation,which fraction may further be diluted in a suitable buffer.

The volatile fraction of the invention may be mixed with water to givethe food/feed additive or formulation of the invention. The food/feedadditive or formulation of the invention may also be prepared by mixingmore than one plant volatile fraction obtained by the process of theinvention. This mixture may be further mixed with water.

The volatile fractions may be prepared from may any suitable fruit,vegetable, leaf, stem or root of a plant. The plant can be, for example,apple, citrus fruit, soy bean, beet, garlic, cabbage or alfalfa, or aherb such as parsley, mint or dill. It should be noted that whenappropriate, the formulation may further comprise volatile fractionsfrom apicultural products such as honey or propolis or other beehiveproducts. These volatile fractions may be prepared in the same manner ofthe plant extract volatile fractions.

According to the process of the invention, the distillation step ispreferably carried out under reduced pressure of 5-10 mbar.

As indicated hereinbefore, the food/feed additive or formulation of theinvention may be further combined with at least one probiotic orindustrial agent, to give the-probiotic or industrial compositions ofthe invention. Thus, the process of preparation of the invention mayalso further comprise the step of suspending at least one viableprobiotic or industrial microorganism in the volatile fraction obtainedin said step (d) or in a mixture of such volatile fractions obtained asdescribed above.

According to a particular process of the invention, the probioticmicroorganism suspended may be the novel bacterium of the invention,derived from E. coli and deposited at the ATCC under Deposit No. 202226(and at DSM under deposit No. 12799).

Finally, the invention relates to the use of a volatile fraction (VF) ofa plant extract in the preparation of a food/feed additive, wherein theVF is prepared by steam distillation of said plant extract or fromapiculture product extract such as honey or propolis at a bathtemperature not exceeding 38° C.

An additional aspects of the invention include a process of preparing acomposition including viable microorganisms. The process is effected bysuspending at least one microorganism in a formulation containing atleast one volatile fraction (VF) of a plant extract. The formulation isselected so as to sustain viability of the microorganism(s) for at least2 months at room temperature. The microorganism of choice may beprobiotic or have industrial applicability. Organisms which can bemaintained in a formulation of the present invention include Escherichiaspp., Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp.,Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. and Pseudomonasspp.

As shown in FIG. 1, in another aspect the present invention provides adispenser 10 of microorganisms. Dispenser 10 includes a reservoir 12 anda dispensing mechanism 14 connected thereto. Reservoir 12 houses atleast one microorganism in a formulation containing at least onevolatile fraction (VF) of a plant extract. The formulation is selectedso as to sustain viability of the microorganism(s) for at least 2 monthsat room temperature. Reservoir 12 may be refrigerated, say to about2-20° C., preferably to about 4° C. Dispenser 10 may be an airbornedispenser, so as to allow dispersion of pest control bioagents. Thedispenser may be hand held. In any case, the dispenser may be designedto automatically dispense a predefined volume therefrom, e.g.,periodically. To this end, dispenser 10 may include a programmableor-preset control mechanism 16, valves 18, etc.

Thus, according to another aspect, the present invention provides amethod of biocontrol of an organism which is effected by spreading in anendemic area at least one viable microorganism capable of biocontrollingthe organism. The viable microorganism(s) are present. in a formulationcontaining at least one volatile fraction (VF) of a plant extractselected so as to sustain viability of said at least onemicroorganism(s) for at least 2 months at room temperature.

Still according to another aspect the present invention provides amethod of eliminating an oil pollution which is effected by spreading ina polluted area at least one viable microorganism capable of degradingoil, such as Pseudomonas spp. The viable microorganism(s) is present ina formulation containing at least one volatile fraction (VF) of a plantextract selected so as to sustain viability of said at least onemicroorganism for at least 2 months at room temperature.

Yet according to another aspect the present invention provides a methodof restoring organic fumes degrading microorganism population in abiofilter by dispensing onto the filter at least one viablemicroorganism, such as Pseudomonas spp., Nitrosomonas spp., orArthrobacter spp. capable of degrading organic fumes. The viablemicroorganism(s) is present in a formulation containing at least onevolatile fraction (VF) of a plant extract selected so as to sustainviability of said at least one microorganism for at least 2 months atroom temperature.

Still according to another aspect of the present invention there isprovided a method of preparing a starter for a fermentation process bygrowing a sufficient amount of a starter microorganism, such as,Lactococcus spp., Escherichia spp., Bifidobacterium spp., Lactobacillus,Lactococcus spp. or Pseudomonas spp. and suspending the startermicroorganism in a formulation containing at least one volatile fraction(VF) of a plant extract selected so as to sustain viability of saidstarter microorganism for at least 2 months at room temperature.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Example 1

Preparation of a “Volatile Fraction” of Plant Extracts or Extracts fromApiculture Products

Fresh vegetables obtained from commercial markets were thoroughly washedby tap water, chopped and finely ground in an industrial blender.Distilled water was added to the vegetable biomass at the proportion of3 parts water to 1 part of the vegetable mass by weight and left understirring for at least 2 hours at ambient temperature.

The mixture was then transferred into the evaporating flask of arotatory evaporator and was evaporated at a reduced pressure (5-10 mbar)under such conditions that the temperature of the water bath did notexceed 38° C., and that of the condenser inlet was 2-5° C. About 1 literof the 2.5 kg of plant biomass.

This material could be preserved under refrigeration for at least 12months without loosing its properties, as is further exemplifiedhereinunder.

The same procedure may be carried out, replacing the plant material withapiculture products such as honey or propolis.

Usually, each “volatile fraction” was prepared from a single kind ofvegetable and used in different dilution to prepare different mixturesfor various purposes.

Example 2

Isolation and Growth of the Probiotic Organism E. coli A TCC Deposit No.202226

The probiotic organism E. coli (deposited at the ATCC under No. 202226on May 3, 1999 and at the DSM under No. 12799 on May 4, 1999) wasisolated from E. coli M-17 by sequential transfer of isolates initiatingfrom long term (2 months) survivors preserved in a formulationcontaining volatile fractions of plant derived material as is describedunder Example 1 above at 37° C. and selecting for isolates capable ofcompetition with pathogens as is further detailed below. The probioticorganism E. coli (deposited at the ATCC under No. 202226 on May 3, 1999and at the DSM under No. 12799 on May 4, 1999) was grown in thefollowing medium: (NH₄)₂SO₄—5 g/liter, KH₂PO₄—13 g/liter, Na₂HPO₄—13giliter, MgCI₂—3 g/liter, CaCl₂—0.3 g/liter, yeast extract—10 g/liter,Soy peptone—10 g/liter and glucose—5 g/liter. Additional nutrients(yeast extract—1 g/liter, Soy peptone—2.5 g/liter and glucose—90g/liter) were continuously added: following the growth of the culture insuch a way that the glucose concentration in the fermentation broth waskept at a level of 2 g/liter. The pH of the fermentation broth was keptneutral by the continuous addition of 4N NH₄OH. Culturing was carriedout at 30° C. in a standard fermentation vessel with aeration of 0.5 vvmfor 16 hours, when the growth became confluent. This procedure resultedin 10¹⁰-10¹¹ cells/ml. The E. coli cells were harvested bycentrifugation, resuspended in saline and re-precipitated. The microbialbiomass could be kept in saline in the refrigerator for 48 hours withoutloosing viability.

Example 3

Preparation of Food/feed Additive Formulation for the AcceleratedIncrease of Body Weight in Mammals and Avians

The mixture contained volatile fractions of: alfalfa—50 ml/liter, soybeans—200 ml/liter, beet—25 ml/liter and dill—100 ml/liter, prepared asdescribed under Example 1 above. The balance was made up by thedistilled water.

Example 4

Preparation of a Food Supplement for the Normalization of the Functionof the Human GI Tract.

The mixture contained volatile fractions of: alfalfa—50 ml/liter, soybeans—10 ml/liter, beet—50 ml/liter, dill—50 ml/liter, mint—20 ml/liter,parsley—20 ml/liter and cabbage—10 ml/liter, prepared as described underExample 1 above. The balance was made up by distilled water. E. coli(ATCC 202226) cells (Example 2) were suspended in the mixture at a cellconcentration of 10 cells/ml. NaCi (e.g., 4-10 g/liter) may beoptionally added for the improvement of taste.

Example 5

Preparation of a Feed Additive Formulation for the Prophylaxis of GIInfections in Mammals and Avians

The mixture contained volatile fractions of: alfalfa—50—ml/liter, soybeans—10 ml/liter, beet—50 mi/liter, dill—150 ml/liter and mint—10ml/liter, prepared as described under Example 1 above. The balance wasmade by distilled water. E. coli (ATCC 202226) cells (Example 2) weresuspended in the mixture at a cell concentration of 10⁷ cells/ml. NaCl(4-10 g/liter) may be optionally added for the improvement of the taste.

Example 6

Antagonistic Activity of E. coli (ATCC 202226) Against SalmonellaTyphymirium (ATCC 14028)

Petri plates containing Modified Brilliant Green Agar, a selectivegrowth medium for Salmonella, were inoculated with S. typhimirium. A 9mm diameter well was made in the agar. A volume of the food supplement(Example 4) was deposited in each well, and the plates were incubatedfor 24 hours at 35° C. The same was repeated, but instead of the foodsupplement, the fluid obtained by its filtration through amicrobiological filter membrane (pore size of 0.45 μm) was deposited inthe well.

Around each well containing the feed additive an inhibition zone (10-17mm) devoid of S. typhimirium colonies was observed. No inhibition zonewas observed around the wells containing the filtrate free of theprobiotic organism.

Example 7

Antagonistic Activity of E. coli A TCC 202226 as well as of its ParentStrain E. coli M-17 Against Shigella sps.

Cultures of S. flexneri, S. sonnei, E. coli (ATCC 202226) and M-17 weregrown separately on a Nutrient Agar for 18-20 hours at 37° C. All thecultures were harvested in saline and diluted to an optical density of10 Klett units. Aliquots of the diluted cultures of Shigella species (1ml) alone or in combination with the diluted culture of E. coli (ATCC202226) (1 ml) were seeded in ventilation-cup test tubes containingNutrient Broth (5 ml). The tubes were incubated for 24 hours at 37° C.The number of colony-forming units (CFU) of the pathogens and of E. coli(ATCC 202226) was determined by plating the cultures on Nutrient agar.The CFU numbers of two Shigella species in the pure culture and in mixedcultures with both probiotic E. coli species are shown in Table 1 below.

TABLE 1 Growth of Shigella CFU/ml Probiotic Organism S. flexneri S.sonnel E. coli ATCC 202226 <5 × 10⁴  <5 × 10⁴  E. coli M-17 1 × 10⁶ 2 ×10⁶ Pure Shigella culture 6 × 10⁶ 2 × 10⁶

Example 8

Application of the Food Supplement (Example 4) and of Colibacterin (DryFormulation of E. coli M-17) in Hospitalized Gastroenteritis Patients

A group of 60 patients that developed gastroenteritis followinghospitalization was randomly divided into 3 sub-groups with a similardistribution of sex, age and the severeness of gasroenteritis symptoms.All patients received normal supportive treatment, includingrehydration, vitamins, etc. The severity of condition did not requiretreatment with antibiotics. The first group of 20 patients received 10ml of the food supplement (Example 5) 3 times daily 30 minutes beforethe meals for 7 days. The second group of the same number receivedColibacterin (Colibacterinum siccum) as recommended by the producer (onedose twice a day 30 minutes before the meal) and the third groupreceived no supplement at all.

The onset of normalization (in days) of the symptoms of gastroenteritisin all groups were recorded and are shown in Table 2 below.

TABLE 2 Onset of the normalization of the symptom, days Food Symptomssupplement n Control Body 2.8 ± 0.2 2.7 ± 0.2 3.7 ± 0.2 temperatureIntoxication 2.5 ± 0.1 3.6 ± 0.1 4.6 ± 0.1 Abdominal pain 3.3 ± 0.2 5.1± 0.2 6.1 ± 0.2 Diarrhea 1.8 ± 0.2 3.4 ± 0.2 4.4 ± 0.2

Colibacterin (Colibacterinum siccum) is the freeze-dried preparation oflive E. coli M-17 produced by BIOMED Ltd., Moscow, Russia, andrecommended for use against diarrhea [Vidal Handbook: Pharrnaceuticalpreparations in Russia (N.B. Nikolaeva, B. P. Alperovich and V. N.Sovinov, Eds.) AstraPharmService, 1997, Moscow, p. 2751].

Example 9

Application of the Food Supplement (Example 4) in Patients with Acute GIInfections

Patients with severe GI infections of various etiologies: salmonellosis,escherichiosis, shigellosis, staphylococcal infections and foodintoxications of unknown etiology were included in the study. In allpatients the hospitalization was indicated by an acute onset of thedisease and appearance of acute gastroenteritis. The total of 186patients were treated with the food supplement and a similar group of102 patients received standard treatment.

The onset of normalization (in days) of the symptoms of gastroenteritisin all groups were recorded and are shown in Table 3 below.

TABLE 3 Onset of normalization, days Symptoms Study group Control Fever1.2 ± 0.3 2.7 ± 0.3 Weakness 1.6 ± 0.2 2.9 ± 0.3 Abdominal 1.5 ± 0.2 2.4± 0.3 pain Diarrhea 2.3 ± 0.2 4.7 ± 0.4 Days in bed 4.8 ± 0.3 6.7 ± 0.4

In a separate study a group of 30 patients with intestinal typhoid wastreated with the food supplement. In 80% of patients the symptoms ofdisease disappeared within 3 days. Only in 3 cases the supplementtreatment was stopped due to the development of more severe chroniccolitis.

Example 10

Applications of the Food Supplement (Example 4) in Patients with GIDisorders Caused by Antibiotics

Patients with severe GI disorders were divided in 2 groups. Group Icontained 48 patients with peptic ulcer disease who developed GIdisorders after antibiotic treatment against H. pylori. Group IIcontained 22 patients that developed GI disorders following antibiotictreatment of pneumonia.

The food supplement (5 ml) was given 3 times a day before meals for 7days. In both groups symptoms of diarrhea disappeared in 2-3 days in allthe patients. After the administration of the supplement, completenormalization of intestinal microflora was observed in 84.5% of thepatients. It was demonstrated by a dramatic increase in lactobacilli andBifidobacteria, reduction on the general count of E. coli, completedisappearance of the hemolytic E. coli and other pathogens such asStaphylococci, Proteus vulgaris and even Candida sps. In the remaining15.5% of the patients, a significant improvement was observed.

Example 11

Application of the Food Supplement (Example 5) in Patients with LateRadiation Enterocolitis

The food supplement (10 ml, 3 times a day for 14 days, 30 minutes beforethe meals) was given to 24 patients with womb, colon and gastric cancerthat developed enterocolitis following radiation therapy or acombination of radiation and chemotherapy.

Prior to the use of the food supplement, all patients complained aboutfrequent and painful desire to defecate, liquid stool (4-12 times a day)appearance of mucous (9 cases) and blood (3 cases) in the stool.

Two or three days after ingesting the supplement, all the patients notedthe lessening of pain and reduction in the number of defecations; thestool had a normal appearance. Four to five days later the diarrhealsyndrome was gone, the appearance of blood and mucus ceased. The bloodanalysis showed a strong improvement in blood indicators.

In the control group of a similar size the symptoms persisted.

Example 12

Application of the Food Supplement (Example 4) in AIDS Patients

Patients suffering from AIDS frequently develop chronic diarrhea. Agroup of such patients was given 10 ml of the food supplement (Example5) 3 times a day 30 minutes before the meals for 20 days. The controlgroup received no supplement. The results of the treatment are shown inTable 4 below.

TABLE 4 Parameter Food supplement Control No. of patients 30 20 Averageage, years 38 ± 1  36 ± 2  Daily defecation frequency: at the onset ofthe Exp. 3.4 ± 0.3 3.6 ± 0.3 at the end of the Exp. 1.1 ± 0.1 3.2 ± 0.3one month after the Exp. 1.5 ± 0.2 3.7 ± 0.3 Av. onset of normalization6.0 ± 0.7 remained abnormal

Patients receiving the food supplement showed normalization ofintestinal microflora: reduction in general number of coliforms,disappearance of the hemolytic E. coli, increase in the numbers ofLactobacilli and Bifidobacteria, reduction in Candida sps.

Example 13

Application of the Food/feed Additive (Example 3) for Accelerated WeightIncrease in Healthy Piglets

Healthy piglets were administrated 3 ml per os of the food/feed additiveper piglet per day until weaning. The group receiving the feed additivegained weight at weaning on the average 1.0 kg per piglet more than thecontrol group.

Example 14

Application of the Feed Additive (Example 5) in Healthy Piglets

Several hundreds healthy piglets were administrated 3 ml per os of thefeed additive per piglet on the first and third day after delivery andat weaning. Mortality was cut down by 50% compared with the controlgroup I-receiving standard prophylactic treatment with antibiotics. Thegroup receiving the feed additive gained weight at weaning on theaverage 0.39 kg per piglet more than the control group. When the feedadditive and antibiotic treatment were compared in the same litter, theweight gain in the piglets obtaining the feed additive was found higherby 2.4 kg than in the control.

Example 15

Application of the Feed Additive (Example 4) in Piglets Showing Diarrhea

Several hundred piglets showing diarrhea were given daily 5 ml per os ofthe feed additive per piglet. The control group of the same size wastreated with antibiotics: advocin, gentiamycin, amoxicillin. Thesymptoms of diarrhea in the group receiving the feed additivedisappeared within 1-2 days. No mortality was observed, and pigletsdeveloped normally. Antibiotics stopped diarrhea in the great majorityof piglets but the piglets remained stunted in their development.

About 70 retarded piglets, that received antibiotic treatment againstdiarrhea for a week, and generally considered lost, were given the feedadditive for three days. All but two survived.

Example 16

Application of the Feed Additive (Example 4) in Healthy Calves

Day-old healthy calves were administrated 5 ml of the feed additive aday in milk during 7 days. More than 95% of the calves did not developeddiarrhea until they were 14 days old, when a few cases positive forRotavirus were diagnosed. Normally, the incidence of diarrhea in thisfarm is 20-30%.

Example 17

Application of the Feed Additive (Example 6) in Calves Showing Diarrhea

Calves developing diarrhea were given daily 10 ml of the feed additiveper animal with milk during 3-5 days. The symptoms of diarrheadisappeared within 1-2 days in 90-95% of calves. In the remaining 5-10%the diarrhea was caused by virus. These calves were treated withantibiotics with poor results.

Example 18

Application of the Feed Additive (Example 6) in Healthy Lambs and GoatKids

Day-old healthy lambs and goat kids were administrated 3 ml of the feedadditive a day in milk during 7 days. In some cases slight to moderatesigns of diarrhea were observed. These signs usually disappearedspontaneously or were successfully treated with an increased dose of thefeed additive (5 ml). Normally the insidence of diarrhea in these farmsis 50% for baby lambs and for goat kids.

Example 19

Application of the Feed Additive (Example 5) in Lambs and Goat KidsShowing Diarrhea

The feed additive was tested in a herd suffering from pathogenic E. coliinfections. In the year preceding the experiment, about 90 from 120lambs and goat kids died from diarrhea. Treatment with antibiotics wasineffective, since the disease developed suddenly and with fastmortality. Lambs and goat kids developing diarrhea were given daily 5 mlof the feed additive per animal with milk during 3-5 days. A matchinggroup of animals was treated, as normally recommended, with antibiotics.The symptoms of diarrhea disappeared within 1-2 days in about 90% of thelambs and goat kids receiving the feed additive. Their furtherdevelopment appeared normal. The control group receiving antibiotics(gentarnycin). showed much poorer results. Diarrhea in this grouppersisted, calling for repeated treatment with antibiotics. Thedevelopment of the control group was severely retarded.

Example 20

Application of the Feed Additive (Example 5) in Poultry

The feed additive was added to the drinking water with the averageuptake of 0.01 ml of the additive per day per chick during the breedingperiod (42-49 days). An increase of 3.2% in weight gain, accompanied by4% improvement in food conversion was noted in controlled trials inbroilers.

Excellent results were also obtained with turkeys of age 1 day to 6weeks. Each bird received 0.01 ml of the additive per day. A weight gainof over 10% was observed, along with reduced mortality rate. Birds whichstill exhibited diarrhea, were treated with 0.1 ml per day of the foodadditive, without any treatment by antibiotics, and showed betterrecovery from the control birds which were treated with onlyantibiotics.

Example 21

Application of the Feed Additive (Example 5) in Dogs and Cats

Application of the feed additives in puppies resulted in cessation ofthe symptoms of diarrhea within 24-48 hours.

Example 22

Application of the Feed Additive (Example 5) in Mice; Evidence forImmunostimulation

Two-weeks-old C571B1 mice were divided into 4 groups of 10 animals ineach. Mice were deprived water from 4:00 pm to 8:00 am and then given 15ml of either water (groups 1 & 3) or the feed additive (groups 2 & 4).This treatment was repeated for 2 weeks. At the end of this treatmentmice from groups 3 and 4 were subjected to laparotomy under ethernarcosis. A cut of 1.5 cm was made in the abdominal wall. Irritation ofintestine was carried out by a dry gause. All the mice were sacrificed24 hours after laparotomy; spleens were excised. Splenocytes wereisolated and cultured on RPMI medium for 48-72 hours in the presence ofphytohemagglutinin (PHA). In the group treated with the feed additivethe proliferative activity of splenocytes reduced by laparotomy wasrecovered.

Splenocytes proliferation induced by PHA:

1. Control group/water 3060±290

2. Control group/water/laparotomy 2120±300

3. Test group/BioCocktail 2740±370

4. Test group/BioCocktail/laparotomy 3040±520

Example 23

Application of the Food Additive (Example 4) in Human; Evidence forImmunostimulation

A number of patients were operated for malignant tumors of womb, cervix,stomach, and intestine. The gynecological patients (20 patients)received. follow-up radiotherapy; the abdominal patients (30 patients)received chemotherapy. About half of the patients received the foodadditive twice daily for the duration of therapy. In all patientsreceiving the food additive there was observed a significantimmunostimulation, as expressed in:

Gynecological tumors Abdominal tumors Food Food Control Additive ControlAdditive T-lymphocytes 0.03 ± 0.01 0.10 ± 0.03 1.0 ± 0.1 1.6 ± 0.2(CD3+, HLA−, DR+) Lymphocyte blast transformation a. spontaneous 530 ±110 880 ± 80  700 ± 120 1070 ± 100  b. PHA-     induced 1600 ± 360  5720± 2100 4400 ± 1340 13370 ± 3720  Phagocytes No. 60.7 ± 2.9  76.5 ± 2.2 57.7 ± 1.5  74.2 ± 1.9  (%)

Example 24

Bacteria Preservation Effect of the Formulation

E. coli cells were dispersed in a formulation prepared as describedunder Example 1 above and was maintained for 6 months at room 20,temperature. Both at the beginning and at the end of the 6 monthsperiod, samples derived from the dispersion were seeded at appropriateconsecutive dilutions on nutrient broth agar plates. The number ofcolony forming units was determined for both samples. The concentrationdetermined was 1×10⁷ in both cases, showing the effect of theformulation in preserving microorganisms. The above experiment wasperformed in parallel with Pseudomonas putida, yielding the sameresults.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A formulation comprising at least one volatilefraction (VF) of a plant extract, wherein said plant extract is obtainedby water extraction and said volatile fraction being prepared by steamdistillation of said plant extract under a pressure lower thanatmospheric pressure and at a bath temperature not exceeding 38° C. 2.The formulation of claim 1, wherein said plant extract is obtained froma plant organ selected from the group consisting of leaves, stems, rootsand fruit.
 3. The formulation of claim 1, wherein said plant extract isobtained from a plant selected from the group consisting of a vegetableand a herb.
 4. The formulation of claim 3, wherein said vegetable isselected from the group consisting of soy bean, alfalfa, garlic, beetand cabbage.
 5. The formulation of claim 3, wherein said herb isselected from the group consisting of parsley, mint and dill.
 6. Theformulation of claim 1, further comprising a beehive product.
 7. Theformulation of claim 6, wherein said beehive product is propolis.